Hydrogel for resisting inflammation and promoting repair of uterine cavity as well as preparation method and application of hydrogel

Abstract

The invention relates to an anti-inflammatory and repair-promoting hydrogel for a uterine cavity as well as a preparation method and application of the anti-inflammatory and repair-promoting hydrogel, and relates to the technical field of biological medicines. The uterine cavity anti-inflammatory and repair-promoting hydrogel is prepared from the following raw materials: an anti-inflammatory component, a biomacromolecule with active ester and an amino high-molecular polymer. The hydrogel for resisting inflammation and promoting repair of the uterine cavity, which is prepared by the invention, has good biological safety, injectability and adhesiveness with tissues; in intrauterine adhesion prevention treatment, a three-dimensional network structure formed by crosslinking of the biomacromolecules with the active ester and the amino high-molecular polymer plays a physical isolation role, the anti-inflammatory component plays an anti-inflammatory role by inhibiting expression of an inflammation signal channel, and the biomacromolecules with the active ester and the amino high-molecular polymer achieve a synergistic treatment effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a uterine cavity anti-inflammatory and repair-promoting hydrogel, a preparation method and application thereof. BACKGROUND

[0002] The female uterus plays an important role in physiological and reproductive functions. The normal endometrium has periodic changes and regenerative capacity. However, curettage, abortion surgery, hysteroscopy, endometrial infection, pelvic radiotherapy, hormone imbalance, congenital uterine malformation, etc. can cause endometrial damage, thereby activating the immune system and recruiting neutrophils and macrophages. Neutrophils release reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), which destroy normal tissue repair. M1-type macrophages secrete pro-inflammatory factors (TNF-α, IL-6), promoting fibrosis; M2-type macrophages over-activate and secrete TGF-β, stimulating fibroblast proliferation. Abnormal endometrial repair and enhanced fibrosis eventually lead to intrauterine adhesions (IUA). Intrauterine adhesions (IUA) are also known as Asherman's syndrome, which is clinically manifested as menstrual abnormalities, infertility, repeated miscarriage, pelvic pain and other problems.

[0003] At present, hysteroscopic surgery is the main treatment method, and intrauterine devices (IUDs), balloon stents or sodium hyaluronate are placed after surgery to prevent the uterine wall from adhering again. In addition, oral estrogen or traditional Chinese medicine, and injection of antibiotics and other drugs are also used to treat intrauterine adhesions. However, physical barriers and drug therapy have limited effects and side effects.

[0004] As a biological material, hydrogel has good biological safety and biodegradability, and can be used as a physical barrier in the treatment of intrauterine adhesions. Some components such as hyaluronic acid, chitosan and polypeptides can promote endometrial repair, and there are cases of using hydrogel for postoperative anti-adhesion. However, the existing hydrogel has problems such as poor adhesion and mismatched degradation rate. Therefore, the present application provides a uterine cavity anti-inflammatory and repair-promoting hydrogel to solve the problems of the prior art and improve the treatment effect. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a uterine cavity anti-inflammatory and repair-promoting hydrogel, a preparation method and application thereof. The purpose is to provide a composite hydrogel with strong adhesion, controllable degradation, good biological safety, anti-inflammatory and repair-promoting properties.

[0006] The technical solution of the present application to solve the above technical problems is as follows:

[0007] In a first aspect, the uterine cavity anti-inflammatory and repair-promoting hydrogel is prepared from an anti-inflammatory component, a bioactive ester-containing biomacromolecule, and an amino high polymer, wherein the anti-inflammatory component accounts for 0.1-8% of the mass of the uterine cavity anti-inflammatory and repair-promoting hydrogel; the bioactive ester-containing biomacromolecule accounts for 0.5-32% of the mass of the uterine cavity anti-inflammatory and repair-promoting hydrogel; and the amino high polymer accounts for 1-20% of the mass of the uterine cavity anti-inflammatory and repair-promoting hydrogel.

[0008] The bioactive ester-containing biomacromolecule and the amino high polymer are crosslinked into a three-dimensional network structure; and the anti-inflammatory component plays an auxiliary role in the repair of uterine cavity adhesion.

[0009] The uterine cavity anti-inflammatory and repair-promoting hydrogel has the following advantages:

[0010] (1) The uterine cavity anti-inflammatory and repair-promoting hydrogel has good biological safety, injectability, and adhesion to tissues; the bioactive ester-containing biomacromolecule and the amino high polymer can promote the repair of the endometrium; and the anti-inflammatory component can inhibit inflammatory signaling pathways (such as NF-κB and MAPK) and down-regulate the expression of pro-inflammatory factors to exert anti-inflammatory, antioxidant, and anti-fibrotic functions.

[0011] (2) The uterine cavity anti-inflammatory and repair-promoting hydrogel has good toughness and adhesion, and can adhere to the inner surface of the uterine cavity without falling off when applied to the uterine cavity wound; the hydrogel is soft and its shape can adapt to the shape of the cavity, thereby preventing damage to the uterine wall; the hydrogel can physically isolate the uterine wall to fundamentally prevent the occurrence of uterine cavity adhesion and reduce the incidence of uterine cavity adhesion; the hydrogel can rapidly gel in the body within 10-120 seconds, gradually degrade into a water solution with good flowability within 4-7 days, and be discharged from the body.

[0012] Based on the above technical solution, the uterine cavity anti-inflammatory and repair-promoting hydrogel can be further improved as follows.

[0013] Further, the anti-inflammatory component includes at least one of tetramethylpyrazine, paeoniflorin, glycyrrhizic acid, chlorogenic acid, curcumin, a glucocorticoid (hydrocortisone, prednisone, methylprednisolone, dexamethasone, betamethasone), adalimumab, etanercept, tocilizumab, rituximab, tofacitinib, estrogen, pentoxifylline, and a non-steroidal anti-inflammatory drug (ibuprofen, naproxen, diclofenac, indomethacin, aspirin, celecoxib, meloxicam, and acetaminophen).

[0014] The beneficial effect of the above further scheme is that the anti-inflammatory component can play the functions of anti-inflammation, anti-oxidation and anti-fibrosis by inhibiting inflammatory signaling pathways (such as NF-κB, MAPK, etc.) and down-regulating the expression of pro-inflammatory factors, thereby enhancing the effect of the prepared hydrogel on preventing intrauterine adhesion.

[0015] Further, the bio-macromolecule with active ester includes at least one of hyaluronic acid (5-1000 kDa), fish collagen peptide, polyglutamic acid, whey protein peptide and soybean peptide.

[0016] The beneficial effect of the above further scheme is that the bio-macromolecule with active ester such as hyaluronic acid (5-1000 kDa), fish collagen peptide, polyglutamic acid, whey protein peptide and soybean peptide has a higher cross-linking degree of the stereo structure formed when reacting with the amino high molecular polymer, better continuity and better biological safety.

[0017] Further, the amino high molecular polymer includes at least one of gelatin, polypeptide, chitosan, carboxymethyl chitosan, 2-arm polyethylene glycol amino, 4-arm polyethylene glycol amino and 8-arm polyethylene glycol amino. polyetherimide.

[0018] The beneficial effect of the above further scheme is that the amino high molecular polymer is degradable and has good biological safety, and can react with the bio-macromolecule with active ester at room temperature; in addition, hyaluronic acid, polypeptide, chitosan and carboxymethyl chitosan also have the abilities of anti-inflammation and promoting repair, and can enhance the effect of the hydrogel on intrauterine adhesion repair.

[0019] Further, the total solid content of the intrauterine anti-inflammatory and repair-promoting hydrogel is 1%-20%.

[0020] The beneficial effect of the above further scheme is that the hydrogel in this range has better adhesion, flexibility and injectability.

[0021] In a second aspect, a preparation method of an intrauterine anti-inflammatory and repair-promoting hydrogel includes the following steps:

[0022] The anti-inflammatory component and the bio-macromolecule with active ester are compounded and dissolved in a first solvent to obtain component A; an amino high molecular polymer is dissolved in a second solvent to obtain component B; and the component A and the component B are mixed.

[0023] The beneficial effect of the above scheme is that the preparation method of the intrauterine anti-adhesion hydrogel of the present application is simple, stable and plays an auxiliary role in the human self-repair process.

[0024] Further, the anti-inflammatory component and the bio-macromolecule with active ester are dissolved in the first solvent by stirring, or the amino high molecular polymer is dissolved in the second solvent by stirring.

[0025] Further, the mass concentration of the anti-inflammatory component in the component A is 1 mg / mL-160 mg / mL.

[0026] The mass concentration of the bio-macromolecule with active ester in the component A is 5 mg / mL-640 mg / mL.

[0027] The mass concentration of the amino high molecular polymer in the component B is 10 mg / mL-250 mg / mL.

[0028] The beneficial effect of the above further scheme is that the bio-macromolecule with active ester and the amino high molecular polymer are moderately cross-linked at a specific concentration to form a hydrogel with adhesion, flexibility and continuous flow.

[0029] Further, the volume ratio of the component A to the component B is 1:1-8.

[0030] Further, the first solvent includes at least one of ultrapure water, deionized water, distilled water, phosphate buffer solution, bicarbonate buffer solution, and borate buffer solution.

[0031] The second solvent includes at least one of ultrapure water, deionized water, distilled water, phosphate buffer solution, bicarbonate buffer solution, and borate buffer solution.

[0032] In a third aspect, the application provides a use of the uterine cavity anti-inflammatory and repair-promoting hydrogel in the preparation of a product for preventing uterine cavity adhesion and / or promoting endometrial repair.

[0033] The uterine cavity anti-inflammatory and repair-promoting hydrogel of the present application is used by mixing the components in a mixing syringe, and then injecting the hydrogel through the extension tube of the syringe to adhere to the surface of the uterine cavity wall wound, so as to achieve the effect of preventing adhesion by isolating the contact of the uterine cavity wall wound and promoting endometrial repair. Specifically, after the uterine cavity surgery is completed, the flowing gel precursor liquid (the mixed liquid of component A and component B before cross-linking) is injected into the uterine cavity by using the syringe; the precursor liquid injected into the uterine cavity forms a flexible gel state at body temperature, which can not only support the mechanical matrix of the uterine cavity, but also adjust its shape according to different shapes and sizes of the uterine cavity; after being injected into the uterine cavity, the cross-linking reaction occurs within 10-120 seconds to form an anti-adhesion gel; since the cross-linking reaction is between active ester and amino group, the amino active ester formed by the reaction slowly degrades in the uterine cavity, and finally the anti-adhesion gel gradually changes into a flowing liquid and is naturally discharged out of the body under physiological conditions. This phase transition self-adaptive uterine cavity anti-adhesion liquid can adjust cell behavior through stress relaxation and help promote tissue regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The hydrogel prepared in Example 1 of the present application is shown in the figure after being dyed with brilliant green;

[0035] Figure 2 Shear lap joint test results of the hydrogels prepared in Example 1, Example 7, Example 8 and Example 9 of the present application; wherein (A) is a test photo, and (B) is a test result;

[0036] Figure 3 In-vitro degradation test result graph of the hydrogels prepared in Example 3 and Example 7 of the present application;

[0037] Figure 4 In-vivo degradation test result graph of the hydrogels prepared in Example 8 and Example 9 of the present application injected into the uterine cavity of SD rats;

[0038] Figure 5 Sensory evaluation of the hydrogel prepared in Example 2 of the present application;

[0039] Figure 6 Water flushing experiment of the hydrogel prepared in Example 2 of the present application;

[0040] Figure 7 Cytotoxicity of the hydrogels of Example 7 and Example 9 under different concentrations of leaching liquor respectively incubated for 24h and 48h.

[0041] Figure 8 Influence of the hydrogels of Example 1, Example 2 and Example 8 on cell inflammation. DETAILED DESCRIPTION

[0042] The principles and features of the present application are described below, and the examples are used to explain the present application, but not to limit the scope of the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0043] Source of materials and reagents:

[0044] Tetramethylpyrazine, glycyrrhizic acid, chlorogenic acid, curcumin, lipopolysaccharide were purchased from Solabio; Paeoniflorin, water-soluble dexamethasone were purchased from MCE; 2-Arm PEG-NH2, 4-Arm PEG-NH2, 8-Arm PEG-NH2 were purchased from Xiamen Sainobor Bioscience; Gelatin (derived from animal skin, bone or tendon) was purchased from Sigma; Polypeptide was purchased from Aladdin; Hyaluronic acid (5-1000kDa), chitosan, whey protein peptide, polyglutamic acid were purchased from Macklin; Carboxymethyl chitosan was purchased from Hydrafine Biological; Rats were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.; RAW 264.7 cell and L929 cell lines were from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences; Mouse interleukin 6 (IL-6) enzyme-linked immunosorbent assay kit and mouse tumor necrosis factor alpha (TNF-α) enzyme-linked immunosorbent assay kit were purchased from Wuhan Elyra Biotech Co., Ltd.

[0045] Example 1: Comparative Example

[0046] This example relates to a method for preparing a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 50 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, dissolving in 1.95 g of deionized water, marked as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and dissolving under stirring at 50-60℃, marked as component B. Component A and component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of component A and 4.0 mL of component B are sucked into the mixed syringe, and the two components are mixed well by the two-way channel of the syringe, and then the mixture is injected out. After waiting for 1 min, the uterine cavity anti-adhesion hydrogel is obtained. The hydrogel prepared in this example is shown in Figure 1 Figure 1 The sample in

[0047] Example 2

[0048] ​The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 2 mg of water-soluble dexamethasone anti-inflammatory component and 50 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, adding 1.948 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. Component A and component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of component A and 4.0 mL of component B are sucked into the mixed syringe, the two components are fully mixed by the two channels of the syringe, and the mixed solution is injected out to wait for 1 min to obtain the uterine cavity anti-adhesion hydrogel.

[0049] Example 3

[0050] The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 2 mg of water-soluble dexamethasone anti-inflammatory component and 50 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, adding 1.948 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. Component A and component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of component A and 4.0 mL of component B are sucked into the mixed syringe, the two components are fully mixed by the two channels of the syringe, and the mixed solution is injected out to wait for 1 min to obtain the uterine cavity anti-adhesion hydrogel.

[0051] Example 4

[0052] The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 2 mg of water-soluble dexamethasone anti-inflammatory component and 50 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, adding 1.948 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. Component A and component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of component A and 4.0 mL of component B are sucked into the mixed syringe, the two components are fully mixed by the two channels of the syringe, and the mixed solution is injected out to wait for 1 min to obtain the uterine cavity anti-adhesion hydrogel.

[0053] Example 5: Comparative Example

[0054] The embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, and the method comprises the following steps: accurately weighing 140 mg of fish collagen peptide active ester (FCP-NHS) in a centrifugal tube, adding 1.86 g of deionized water to dissolve, and marking as component A; accurately weighing 300 mg of carboxymethyl chitosan in a sample bottle, adding 9.70 g of alkaline deionized water, and stirring and dissolving at 50-60 DEG C, and marking as component B. The component A and the component B are respectively injected into a 1:1 mixed syringe, 1.0 mL of the component A and 1.0 mL of the component B are sucked into the mixed syringe, the two components are fully mixed and injected out through a two-channel syringe, and the uterine cavity anti-adhesion hydrogel can be obtained after waiting for 2 min.

[0055] Example 6: Comparative example

[0056] The embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, and the method comprises the following steps: accurately weighing 40 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifugal tube, adding 1.96 g of deionized water, and stirring and dissolving the hyaluronic acid active ester, and marking as component A; accurately weighing 200 mg of eight-arm polyethylene glycol amino (8-Arm PEG-NH2) in a centrifugal tube, adding 1.80 g of deionized water, and ultrasonically dissolving to fully dissolve the polyethylene glycol, and marking as component B. 1.0 mL of the component A and 1.0 mL of the component B are respectively sucked into a 1:1 mixed syringe, the two components are fully mixed and injected out through a two-channel syringe, and the uterine cavity anti-adhesion hydrogel can be obtained after waiting for 1 min.

[0057] Example 7: Comparative example

[0058] The embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, and the method comprises the following steps: accurately weighing 40 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifugal tube, adding 1.96 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60 DEG C, and marking as component B. The component A and the component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of the component A and 4.0 mL of the component B are sucked into the mixed syringe, the two components are fully mixed and injected out through a two-channel syringe, and the uterine cavity anti-adhesion hydrogel can be obtained after waiting for 1 min.

[0059] Example 8

[0060] The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 2 mg of anti-inflammatory ingredient curcumin and 20 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, adding 1.978 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. The component A and the component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of the component A and 4.0 mL of the component B are sucked into the mixed syringe, the two components are fully mixed and injected out through two channels of the syringe, and the uterine cavity anti-adhesion hydrogel can be obtained after waiting for 1 min.

[0061] Example 9

[0062] The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 2 mg of anti-inflammatory ingredient curcumin and 20 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, adding 1.978 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. The component A and the component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of the component A and 4.0 mL of the component B are sucked into the mixed syringe, the two components are fully mixed and injected out through two channels of the syringe, and the uterine cavity anti-adhesion hydrogel can be obtained after waiting for 1 min.

[0063] Example 10: Comparative example

[0064] The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 2 mg of anti-inflammatory ingredient curcumin and 20 mg of hyaluronic acid active ester (HA-NHS 5kDa) in a centrifuge tube, adding 1.978 g of deionized water to dissolve, and marking as component A; accurately weighing 400 mg of carboxymethyl chitosan in a sample bottle, adding 9.60 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. The component A and the component B are respectively injected into a 1:4 mixed syringe, 1.0 mL of the component A and 4.0 mL of the component B are sucked into the mixed syringe, the two components are fully mixed and injected out through two channels of the syringe, and the uterine cavity anti-adhesion hydrogel can be obtained after waiting for 1 min.

[0065] Example 11: Comparative example

[0066] The present embodiment relates to a preparation method of a uterine cavity anti-inflammatory and repair-promoting hydrogel, comprising the following steps: accurately weighing 560 mg of fish collagen peptide active ester (FCP-NHS) in a centrifugal tube, adding 1.44 g of deionized water for ultrasonic dissolution, and marking as component A; accurately weighing 300 mg of carboxymethyl chitosan in a sample bottle, adding 9.70 g of deionized water, and stirring and dissolving at 50-60°C, and marking as component B. The component A and the component B are respectively injected into a 1:4 mixing syringe, 1.0 mL of the component A and 4.0 mL of the component B are sucked into the mixing syringe, and the two components are fully mixed and injected out through a two-way channel of the mixing syringe.

[0067] Test example

[0068] (1) Gelation time.

[0069] The preparation method of the hydrogel in the above-mentioned embodiments 1-9 and embodiments 10-11 is used, the two-way tube of the mixing syringe is used for injection into a glass dish, and the stopwatch is started at the same time, the gelation time is recorded when the gel is formed, and the state of the hydrogel is observed. The experimental results are shown in Table 1.

[0070] Table 1 Gelation time experimental results

[0071]

[0072] As shown in Table 1, the components A and the components B in embodiments 1-9 show a shorter gelation time at a suitable ratio. The mass concentrations of the components A and the components B are adjusted in embodiments 10 and 11 respectively, and the gelation is significantly affected at an unsuitable ratio, showing a longer gelation time or even no gelation.

[0073] (2) Shear lap experiment.

[0074] The preparation method of the hydrogel in embodiments 1, 7, 8 and 9 is used, the hydrogel is injected into the inner surface of a flat and degreased pigskin which is cut into a size of 7 cm long and 1 cm wide, another piece of pigskin is quickly adhered to the hydrogel after injection, and is pressed uniformly. Then the prepared sample is pressed flat and placed in a 4°C refrigerator overnight. Then the electronic tensile testing machine is used to test the sample, as shown in Figure 2 A. The shear lap experiment results are shown in Figure 2 B.

[0075] As can be seen from Figure 2 B, the viscosity of embodiment 7 reaches 20.9 kPa, while the viscosities of embodiments 1, 8 and 9 are similar, and the average viscosity is 13 kPa. The results show that the hydrogels prepared by different proportions of active ester-containing hyaluronic acid and carboxymethyl chitosan have different viscosities, and embodiment 7 shows the largest viscosity.

[0076] (3) In vitro degradation of the hydrogel.

[0077] The method for preparing the hydrogel of Example 3 and Example 7 was used. After the gelation, 80 mg of the hydrogel was taken into a 10 mL centrifuge tube, 10.0 mL of phosphate buffer was added, and the centrifuge tube was placed in a constant temperature shaking water bath at 37°C and at a rotation speed of 100 rpm / min. The in vitro degradation experiment was performed, and the gel weight was recorded after the same volume of phosphate buffer was replaced every certain period of time. The in vitro degradation results are shown in Figure 3 .

[0078] As shown in Figure 3 , the maximum swelling ratio in Example 3 reached 2.3 times, and the hydrogel was not completely degraded at 28 days. The maximum swelling ratio in Example 7 was only 1.4 times, and the hydrogel was basically completely degraded at 25 days. In the in vitro degradation, the hydrogel prepared from the fish collagen peptide active ester showed a higher swelling ratio and a slower degradation rate than the hydrogel prepared from the hyaluronic acid active ester.

[0079] (4) In vivo degradation of the hydrogel in the uterine cavity of SD rats.

[0080] The method for preparing the hydrogel of Example 8 and Example 9 was used. After the 8-week-old SD rats were anesthetized with isoflurane, the abdomen was shaved and disinfected. The abdominal skin was cut transversely at 2-3 cm above the pubic symphysis for about 3 cm, and the tissues were cut layer by layer to enter the abdominal cavity. The Y-shaped uterus of the rat was slowly picked out. A blunt needle was used to enter the rat uterus through the rat vagina, and then a double-end needle of a mixing syringe was used to inject 100 μL of the hydrogel into the uterine cavity of the SD rat through the blunt needle. After observing that the entire uterine cavity was full, the injection was stopped. After the gelation, the uterus was returned to the abdominal cavity, and then the wound was sutured. The degradation of the hydrogel in the uterine cavity of the SD rat was observed. The observed results are shown in Figure 4 .

[0081] It can be concluded from Figure 4 that the hydrogels prepared in Example 8 and Example 9 were degraded only after 4-7 days in the uterine cavity of the SD rat. After the degradation, the appearance and state of the SD rat uterus were almost the same as those of the SD rat without injection of the hydrogel.

[0082] (5) Sensory evaluation and water flushing experiment of the hydrogel.

[0083] The method for preparing the hydrogel of Example 2 was used. The hydrogel was injected into the inner surface of the wet pigskin, and after the gelation, the pigskin was deformed by force. The change in the state of the gel was observed, and the results are shown in Figure 5 . Subsequently, the pigskin with the injected hydrogel was placed under a water faucet, and the change in the gel under the water flow was observed. The results are shown in Figure 6as shown.

[0084] As Figure 5 shown, the hydrogel of Example 2 quickly gelled on the wet pig skin surface and firmly adhered to the pig skin surface, and the gel structure could not be destroyed whether twisted, pulled or pressed, and the hydrogel still adhered to the pig skin surface and showed no signs of falling off. The water flushing experiment is as Figure 6 As shown, under the continuous impact of water flow, the hydrogel of Example 2 did not fall off from the pig skin surface and the surface structure of the gel was not damaged.

[0085] (6) Cytotoxicity experiment.

[0086] Take 400 mg of the intrauterine anti-adhesion hydrogel prepared in Example 7 and Example 9 into a centrifuge tube, irradiate under ultraviolet for 30 min, then add 4 mL of complete medium, and place in a 4°C refrigerator for extraction overnight, then take out the extraction solution for standby. Take L929 cells in logarithmic growth phase, digest, then centrifuge at 1000 rpm for 5 min, then count using a cell counting plate, dilute the cells to 1 x 10 4 cells per well, place the well plate in a cell culture incubator, 4 h after the cells adhere, aspirate the culture medium and add the extraction solution, the concentration of the prepared intrauterine anti-adhesion hydrogel extraction solution is 0 mg / L (control group), 10 mg / mL, 20 mg / mL, 50 mg / mL and 100 mg / mL, then co-incubate for 24 h and 48 h, detect the cell viability by CCK-8 experiment, the results are as Figure 7 shown.

[0087] From Figure 7 It can be concluded that the cell viability is higher than 80% when L929 cells are cultured in the above extraction solutions (0 mg / mL, 10 mg / mL, 20 mg / mL, 50 mg / mL and 100 mg / mL) for 24 h and 48 h, which proves that the intrauterine anti-adhesion hydrogel has good biological safety.

[0088] (7) Cell anti-inflammatory experiment.

[0089] Take 400 mg of the intrauterine anti-adhesion hydrogel prepared in Example 1, Example 2 and Example 8 into a centrifuge tube, irradiate under ultraviolet for 30 min, then add 4 mL of complete medium, and place in a 4°C refrigerator for extraction overnight, then take out the extraction solution for standby. Take RAW 264.7 cells in logarithmic growth phase, digest, then centrifuge at 1000 rpm for 5 min, then count using a cell counting plate, dilute the cells to 1 x 10 6RAW 264.7 cells were cultured in a cell culture incubator. After 4 hours of cell adhesion, the culture medium was removed, and 2 mL of extraction buffer and 1 μg / mL lipopolysaccharide (LPS) were added. The cells were cultured for another 24 hours to induce inflammation in RAW 264.7 cells, thus establishing a cellular inflammation model. The anti-inflammatory capacity of the hydrogel was investigated. ELISA kits were used to detect changes in IL-6 and TNF-α levels in RAW 264.7 cells to reflect the anti-inflammatory capacity. The results are as follows: Figure 8 As shown.

[0090] Depend on Figure 8 It can be concluded that different hydrogels have different inhibitory abilities on the inflammation of RAW 264.7 cells. Figure 8 The results showed a significant increase in the expression of inflammatory factors IL-6 and TNF-α in the LPS group, indicating the successful construction of the cellular inflammation model. After incubation with the hydrogel extracts from Examples 1, 2, and 8, the expression of IL-6 and TNF-α in the hydrogel groups significantly decreased, indicating that all three hydrogels could effectively reduce cellular inflammation levels. The drug-loaded hydrogels from Examples 2 and 8 exhibited further enhanced anti-inflammatory capabilities compared to the hydrogel from Example 1 and the drug-only treatment group, suggesting a synergistic anti-inflammatory effect between the anti-inflammatory drug in the drug-loaded hydrogel and the hydrogel itself. Furthermore, in the treatment of intrauterine injuries, drugs alone cannot exert a long-term effect on the injury site, while the adhesive properties of the hydrogel enable the drug to be released slowly and continuously to treat the injury site. The drug enhances the therapeutic effect of the hydrogel, and the two components complement each other for synergistic treatment.

[0091] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A hydrogel for intrauterine anti-inflammatory and repair-promoting effects, characterized in that, The raw materials of the intrauterine anti-inflammatory and repair-promoting hydrogel include: anti-inflammatory components, biomolecules with active esters, and amino polymers. The anti-inflammatory components account for 0.1%-8% of the mass of the intrauterine anti-inflammatory and repair-promoting hydrogel; the biomolecules with active esters account for 0.5%-32% of the mass of the intrauterine anti-inflammatory and repair-promoting hydrogel; and the amino polymers account for 1%-20% of the mass of the intrauterine anti-inflammatory and repair-promoting hydrogel.

2. The intrauterine anti-inflammatory and repair-promoting hydrogel according to claim 1, characterized in that, The anti-inflammatory components include at least one of the following: tetramethylpyrazine, paeoniflorin, glycyrrhizic acid, chlorogenic acid, curcumin, glucocorticoids, adalimumab, etanercept, tocilizumab, rituximab, tofacitinib, estrogen, pentoxifylline, and nonsteroidal anti-inflammatory drugs.

3. The intrauterine anti-inflammatory and repair-promoting hydrogel according to claim 1, characterized in that, The biomacromolecules containing active esters include at least one of hyaluronic acid, fish collagen peptides, polyglutamic acid, whey protein peptides, and soybean peptides.

4. The intrauterine anti-inflammatory and repair-promoting hydrogel according to claim 1, characterized in that, The amino polymer includes at least one of gelatin, polypeptide, chitosan, carboxymethyl chitosan, 2-arm polyethylene glycol amino, 4-arm polyethylene glycol amino, 8-arm polyethylene glycol amino, and polyetherimide.

5. The intrauterine anti-inflammatory and repair-promoting hydrogel according to any one of claims 1 to 4, characterized in that, The total solids content of the intrauterine anti-inflammatory and repair-promoting hydrogel is 1%-20%.

6. A method for preparing a hydrogel for intrauterine anti-inflammatory and repair-promoting purposes according to any one of claims 1 to 5, characterized in that, Includes the following steps: The anti-inflammatory component is compounded with a biomacromolecule containing an active ester and dissolved in a first solvent to obtain component A; the amino polymer is dissolved in a second solvent to obtain component B; component A and component B are then mixed.

7. The method for preparing a hydrogel for anti-inflammatory and repair-promoting effects in the uterine cavity according to claim 6, characterized in that, The mass concentration of the anti-inflammatory component in component A is 1 mg / mL-160 mg / mL; The mass concentration of the biomacromolecules containing active esters in component A is 5 mg / mL to 640 mg / mL. The mass concentration of the amino polymer in component B is 10 mg / mL to 250 mg / mL.

8. The method for preparing an intrauterine anti-inflammatory and repair-promoting hydrogel according to claim 6, characterized in that, The volume ratio of component A to component B is 1:1-8.

9. The method for preparing an intrauterine anti-inflammatory and repair-promoting hydrogel according to claim 6, characterized in that, The first solvent includes at least one of ultrapure water, deionized water, distilled water, phosphate buffer solution, bicarbonate buffer solution, and borate buffer solution; The second solvent includes at least one of ultrapure water, deionized water, distilled water, phosphate buffer solution, bicarbonate buffer solution, and borate buffer solution.

10. The application of a hydrogel for intrauterine anti-inflammatory and repair-promoting effects, characterized in that, The hydrogel according to any one of claims 1 to 5 is used in the preparation of products for preventing intrauterine adhesions and / or promoting endometrial repair.

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